Aldosterone Impairs Mitochondrial Function in Human Cardiac Fibroblasts via A-Kinase Anchor Protein 12

Figures

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  Figure 1. Aldo modulates AKAP-12 and regulates oxidative stress in adult human cardiac fibroblasts. Aldo effects on AKAP-12 protein expression in human cardiac fibroblasts (A,B). Total mtDNA expression was measured in human cardiac fibroblasts (C). Oxidative markers (D,E) and mitochondrial function markers (F) expressed as a fold change relative to controls in lysate cells from Aldo-treated human cardiac fibroblasts. All conditions were performed at least in triplicate. Histogram bars represent the mean ± SEM of 6 assays. For Western blot experiments, the blots were cropped, protein densitometry was expressed in arbitrary units (AU) once normalized to β-actin. *p < 0.05 vs Control. AKAP-12, A-kinase anchoring protein 12; Aldo, Aldosterone; Spiro, Spironolactone; Total mtDNA, Total mitochondrial DNA; TAC, Total antioxidant capacity; CML, carboxy-methyl-lysine; MDA, malondialdehyde; PGC-1α, peroxisome proliferator-activated receptor-gamma coactivator 1 alpha; PHB, prohibitin.

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  Figure 2. AKAP-12 inhibition modifies mitochondrial function and oxidant status in adult human cardiac fibroblasts. AKAP-12 protein (A) expression was measured in AKAP-12-knock-down human cardiac fibroblast. Total mtDNA expression was quantified by RT-PCR (B). Effects of AKAP-12 knock-down on oxidative markers (C,D). Effects of AKAP-12 knock-down on PGC-1α and PHB protein expressions (E). Histogram bars represent the mean ± SEM of 6 assays. For Western blot experiments, the blots were cropped, protein densitometry was expressed in arbitrary units (AU) once normalized to β-actin. For RT-PCR experiments, data was normalized to HPRT and β-actin for cDNA. *p < 0.05 vs Scramble. AKAP-12, A-kinase anchoring protein 12; Total mtDNA, Total mitochondrial DNA; TAC, Total antioxidant capacity; CML, carboxy-methyl-lysine; MDA, malondialdehyde; PGC-1α, peroxisome proliferator-activated receptor-gamma coactivator 1 alpha; PHB, prohibitin.

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  Figure 3. AKAP-12 activation prevented Aldo effects on mitochondrial function and oxidative stress parameters in adult human cardiac fibroblasts. AKAP-12 protein (A) expression was measured in human cardiac fibroblasts over-expressing AKAP-12. Total mtDNA expression was quantified by RT-PCR (B). Effects of AKAP-12 over-expression on peroxide production (C) and oxidative stress markers (D). Effects of AKAP-12 activation on PGC-1α and PHB protein expressions (E). Histogram bars represent the mean ± SEM of 6 assays. For Western blot experiments the blots were cropped, protein densitometry was expressed in arbitrary units (AU) once normalized to β-actin. For RT-PCR experiments, data was normalized to HPRT and β-actin for cDNA. *p < 0.05 vs Scramble. $p < 0.05 vs Aldo. AKAP-12, A-kinase anchoring protein 12; Total mtDNA, Total mitochondrial DNA; TAC, Total antioxidant capacity; CML, carboxy-methyl-lysine; MDA, malondialdehyde; PGC-1α, peroxisome proliferator-activated receptor-gamma coactivator 1 alpha; PHB, prohibitin.

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  Figure 4. AKAP-12, mitochondrial function and oxidative stress markers in hearts from Aldosterone-salt-treated rats. AKAP-12 immunostaining and protein expression (A,B) in myocardium from Controls, Aldo-salt-treated rats and Aldo-salt + Spiro-treated rats. Total mtDNA expression was measured (C). TAC and peroxide production (D). Oxidative stress markers (E). PGC-1α and PHB expressions (F). PGC-1α immunostaining (G). Magnification of the microphotographs 40x. Histogram bars represent the mean ± SEM of each group of subjects. For Western blot experiments, the blots were cropped, protein densitometry was expressed in arbitrary units (AU) once normalized to β-actin. *p < 0.05 vs Control; $p < 0.05 vs Aldo. AKAP-12, A-kinase anchoring protein 12; Aldo, Aldosterone; Spiro, Spironolactone; Total mtDNA, Total mitochondrial DNA; TAC, Total antioxidant capacity; CML, carboxy-methyl-lysine; MDA, malondialdehyde; PGC-1α, peroxisome proliferator-activated receptor-gamma coactivator 1 alpha; PHB, prohibitin.

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  Figure 5. AKAP-12, mitochondrial function and oxidative stress markers in myocardial biopsies from aortic stenosis patients. AKAP-12 immunostaining and AKAP-12 mRNA levels in myocardial biopsies from AS patients (A,B). Total mtDNA expression was measured (C). PGC-1α and PHB mRNA levels (D) and PGC-1α immunostaining (E) in myocardial biopsies. Immunostaining of Nitrotyrosine and CML (E). Magnification of the microphotographs 40x. Histogram bars represent the mean ± SEM of each group of subjects (Control n = 13 and patients with AS, n = 26) in arbitrary units (AU) normalized to HPRT and β-actin for cDNA. *p < 0.05 vs. control group. AS, aortic stenosis; AKAP-12, A-kinase anchoring protein 12; Total mtDNA, Total mitochondrial DNA; PGC-1α, peroxisome proliferator-activated receptor-gamma coactivator 1 alpha; PHB, prohibitin; CML, carboxy-methyl-lysine.

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  Figure 6. Correlation studies in aortic stenosis patients. AKAP-12 protein expression negatively correlated with serum Aldo (A) and positively correlated with PGC-1α mRNA levels (B) in AS patients. Total mtDNA levels positively correlated with PGC-1α mRNA levels in the whole population (C). AKAP-12, A-kinase anchoring protein 12; Aldo, Aldosterone; PGC-1α, peroxisome proliferator-activated receptor-gamma coactivator 1 alpha.